Abstract/Project Summary Gut leakiness, microbial translocation and systemic inflammation are hallmarks of HIV disease progression. Interestingly, chronic opioid abuse is also well documented to induce gut leakiness and sustained systemic inflammation. HIV patients that use intravenous heroin display accelerated HIV disease progression. Although HIV replication is suppressed in HIV-infected adults on modern ART regimens microbial translocation continues long after peripheral CD4+ T cell restoration. The resulting activation of both the innate and adaptive immune systems in ART treated HIV individuals is reported to be associated with markers of inflammation and is an independent predictor of morbidity and mortality. The role of ART in driving the inflammatory process has not been documented. Thus far, very little is known regarding the underlying mechanisms that contribute to gut leakiness and microbial translocation in either opioid abusers or in HIV patients that are on ART. Increasing number of studies strongly support the concept that the gut microbiota, play a significant role in maintaining gut homeostasis and gut barrier function. Under both homeostatic and disease conditions the microbiota influences immune function in several ways, including the release of metabolites and direct microbial interactions. Although a few studies have correlated the host microbiome in HIV infected patients with the metabolome, the interaction between the ``omics'' level analysis and the gut barrier function in the context of antiretroviral treatment (ART) still remain largely unexplored. There is no data on the interaction of the microbiome-metabolome-gut barrier in HIV patients in the context of opioid abuse. The goal of this study is to develop an integrated pipeline for identifying novel connections between the microbiome-metabolome and gut barrier function following HIV infection and HIV infection in the context of Drug Abuse. We will further investigate if treatment with ART restores homeostasis or exacerbates dysbiosis. Specific Aim 1: a) Identification of unique biomarkers and signature profiles in the gut microbiome that are associated with HIV disease progression in the context of opioid abuse and ART b) Determine if altered signature profiles is associated with metabolic consequences and immune activation. Specific Aim 2. Establish a causal relationship between gut dysbiosis, altered metabolome and barrier disruption following HIV infection and in the context of opioid drug abuse and ART using germ free and antibiotic treated humanized mice. Specific Aim 3. Establish that Probiotics will restore gut homeostasis and delay HIV disease progression in the context of opioid abuse and ART. The results from these studies will allow for the development of a pipeline for new therapeutic strategies to attenuate immune activation and reverse HIV disease progression both in HIV infected patients and in HIV infected drug abusing population.